Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. Liver ischemia and reperfusion injury (IRI) occurs as a consequence of tissue inflammation mediated by host innate TLR4 activation and in the absence of exogenous Ags. However, T cells, particularly CD4 subset, play a critical role in the initiation of liver pro-inflammatory immune response against IR. The question arises how putative Ag-specific T cells regulate TLR4-mediated innate immune response without Ag stimulation? We hypothesize that pro-inflammatory effector memory (EM), rather than na[unreadable]ve, CD4 T cells, promote liver innate immune activation without de novo Ag-specific stimulation via the CD154-CD40 pathway. To mimic the real-life setting, we will utilize a murine model of prolonged "cold" ischemia followed by isogeneic orthotopic liver transplantation (OLT). We will use this model in order to test our hypothesis in two specific aims: (1). To determine the specific CD4 T cell subset and its function in liver IRI. We hypothesize that effector, rather than na[unreadable]ve, CD4 T cell subset function in IRI via CD154 signaling. We will reconstitute otherwise IRIresistant T cell-deficient nude mice with distinct CD4 subset -enriched or -depleted splenocytes, and then assess their ability to trigger IR-induced liver damage in OLTs from syngeneic nude donors. To determine whether Th1 and Th2 type effector CD4 T cells have comparable effects in liver IRI, we will employ purified CD4 T cell subsets from groups of Stat4- vs. Stat 6- KO mice and CD154- vs. IFN-? deficient mice. Finally, a series of in vitro T - macrophage and T - hepatocyte co-culture studies will further identify molecular mechanisms by which T cells function in macrophage activation and hepatocyte apoptosis, the key two events during the course of liver IRI. (2) To determine that the previously activated (effector memory;EM) but not na[unreadable]ve, T cells function in an Agnon- specific fashion to trigger liver IRI. In the absence of exogenous Ag, T cells may become activated by cryptic or altered self-Ags, or by Ag non-specific stimulation, such as cytokines and chemokines. To document that previously activated (EM subset), but not na[unreadable]ve CD4 T cells function in liver IRI in an Ag-independent manner, we will use OT II mice (OVA-specific CD4 TCR transgenic in RAG KO background), in which all T cells are in the na[unreadable]ve state due to their minimal cross-reactivity to self Ags. The susceptibility to liver IRI will be tested in groups of na[unreadable]ve or OVA-immunized OT II mice. Then, naive or activated OT II CD4 T cells will be adoptively transferred into nude mice to recreate IRI in OLTs from syngeneic nude donors. Finally, we will employ cell culture systems to determine whether and how Ag-non-specific stimulation triggers CD4 T cells to express CD154. These interlocked experimental series will provide the final clue as to how Ag-specific T cells regulate tissue inflammation in the absence of specific Ag stimulation during liver IR. Public health Relevance: Liver transplantation is an effective therapy for the treatment of end-stage liver disease. However, ischemia/reperfusion injury remains one of the key limitations. The goal of this project is to increase the organ donor pool by devising novel strategies to prevent liver transplant dysfunction resulting from prolonged ischemia, and ultimately to improve the overall success of liver transplantation.